Process for coating biological pesticides and compositions therefrom

ABSTRACT

A pesticidal composition comprising particles of biological pesticides and particles of durable TiO 2  in an amount sufficient to substantially coat the particles of biological pesticides and process for the preparation thereof are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application was filed under 35 U.S.C. 371 from PCT/US97/17393,internationally filed on Sep. 29,1997, which claims priority from U.S.provisional application No. 60/027,512, filed Oct. 7, 1996.

FIELD OF THE INVENTION

The present invention relates to biological pesticide compositionscomprising particles of biological pesticides coated with durable TiO₂and advantageous processes useful for preparing them.

BACKGROUND OF THE INVENTION

One aspect of the present invention relates to baculoviruses which areknown to have insecticidal activity. Their commercial utility has beenlimited though because, without special formulation, they are readilydeactivated by the UV radiation in sunlight. The incorporation of UVprotectants in a variety of ways is known and some can enhance thestability of these viruses somewhat, however such compositions can becomplicated, expensive to prepare and further improvements in stabilityand compatibility with the virus are needed.

One method of stabilizing the virus is to mix a water soluble UVabsorbing substance with the virus in a spray tank. One such material isa sulfonated copolymer of catechin and leucocyanidin (U.S. Pat. No.4,094,969). Although some degree of stabilization is achieved, there isno bond between the virus and the protecting substance hence rainfall ordew may readily separate them. Most simple tank mixes of ingredientssuffer from this general problem and do not offer sufficient cropprotection due to the lack of UV stability of these viruses.

Microencapsulation using a variety of techniques is another method used.However these systems are limited due to the virus's sensitivity to manyencapsulating precursors and the overall expense of the preparations.Many, owing to their transparency in the UV-A and -B region of sunshine,require an additional light screening agent. Typically the screeningagent is incorporated into the capsule wall such as in U.S. Pat. Nos.4,844,896 and 4,948,586; EP 0 653 158 A1; and WO 96/03041. A majorproblem with these preparations is the lack of a suitable triggeredrelease matrix. In other words, the encapsulated virus is constrained ina matrix which requires some process to occur for it to be released atthe site of action. These release processes typically includehydrolysis, desorption or dispersion and by their nature take time tooccur. Due to the limited residence time of the virus in the insect'smidgut, the site of infection, fast and complete release is necessary toreach the full infective potential of the virus

The use of mineral UV screens has also been described Most do not absorbUV light but rather function by reflecting and refracting UV light.Major exceptions to this generalization are zinc oxide and titaniumdioxide which have strong UV absorption bands. Although titanium dioxide(TiO₂) is claimed in Japan [WPI Acc. #90-228659/30, J Econ. Entomol. 69,731(1976), and UK Patent Appl 2,043,4481] to enhance virus stabilitytoward UV radiation, it is also known that when exposed to ultravioletlight from sunlight it becomes reactive. For example, a sunlightirradiated aqueous suspension TiO₂ produced hydroxyl radicals whichinactivated viruses in waste water treatment [Water Research 29 (1) 95,1995].

It has been found that a modified form of TiO₂, referred to as durableTiO₂ (as illustrated in U.S. Pat. No. 4,125,412, EP 0 654 509 Al),provides a better and easier means of stabilization of baculovirusesthan known stabilizers. In addition, other advantages of the use ofdurable TiO₂ will become apparent hereinafter.

SUMMARY OF THE INVENTION

The present invention pertains to a pesticidal composition comprisingparticles of biological pesticides and particles of durable TiO₂, amodified form of TiO₂, in an amount sufficient to substantially coat theparticles of such biological pesticides; and an advantageous process forthe preparation thereof. More specifically, the present compositionpertains to an insecticidal baculovirus with an improved stabilizer.

The durable TiO₂ can be simply mixed with a biological pesticide,preferably though, durable TiO₂ is substantially coated on the surfaceof the biological pesticide. According to one preferred embodiment ofthe present invention, addition of an aqueous slurry of durable TiO₂ toa stirred aqueous slurry of a baculovirus at neutral pH (or alternatelythe virus to the TiO₂ slurry) causes the durable TiO₂ and thebaculovirus to co-precipitate. Preferably , this material is then spraydried to particles less than 50 micron median volume diameter (mvd).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an enlarged (40,000×) photo of the spray-dried product andshows a single coated virus.

DETAILED DESCRIPTION OF THE INVENTION

Microbial pesticides include the following organisms when they act aspesticides: 1) eucaryotic microorganisms including protozoa algae andfungi; 2) procaryotic microorganisms including bacteria and; 3) viruses.Biological pesticides which are particularly enhanced by the presentinvention are those that are degraded by UV light, thus, quickly losingtheir efficacy. Such UV light degradable pesticides include, but are notlimited to, baculoviruses, granulosis viruses, bacillus thuringensis,Metarhizium spp. and Beauveria spp.

One embodiment of this invention pertains to many different virusesbelonging to more than a dozen families that have been isolated frominsects. Among those, members of the family Baculoviridae are consideredto hold most promise for commercial insect control. More detailedinformation concerning the various aspects of baculoviruses asbiopesticides can be found in the following publications: Ignoffo (968),Viruses-Living Insecticides, Current Topics in Microbiology andimmunology, 42, 129-167; Arif and Jamieson (989), The Baculoviruses,Biocontrol of Plant Diseases, Vol. 1 (K. G. Mukerji & K. L. Garg, eds.);Blissard and Rohrmann (1990), Baculovirus Diversity and MolecularBiology, Annual Review of Entomology 35, 127-155; Adams and Bonami(1991), Atlas of Invertebrate Viruses, CRC Press, Boca Raton; Leisy andvan Beek (1992), Baculoviruses, Possible Alternatives to ChemicalInsecticides, Chemistry & Industry, Apr. 1992, pp. 250-254; Vlak (1993),Genetic engineering of baculoviruses for insect control in: "MolecularApproaches to Fundamental and Applied Entomology" (Oakeshott andWhitten, eds.), Springer Verlag, New York; WO 96/36712.

As pertains to the present invention, the term baculovirus embracesinsecticides containing a nuclear polyhedrosis virus or granulosisvirus. As will be appreciated, in relation to microbial insecticides, ingeneral the microbial pathogens themselves, known as occlusion bodies,are not in practice isolated from the mediums in which they are preparedand processed but can be used in a heterogeneous mixture containing arange of materials from the nutrients to the host's metabolic wasteproducts. Particularly preferred embodiments of this invention embraceinsecticides containing, but not limited to, the nuclear polyhedrosisviruses and granulosis viruses for the bollworm (Helicoverpa zea), thecotton bollworm (H. armigera), the tobacco budworm (Heliothisvirescens), the cabbage looper (Trichoplusia ni), the fall armyworm(Spodoptera frugiperda), the beet armyworm (S. exigua), the cottonleafworm (S. littoralis), the pink bollworm (Pectinophora gossypiella),the Douglas-fir tussock moth (Orgyia pseudotsugata), the spruce budworm(Choristoneura fumiferana), the western spruce budworm (C.occidentalis), the gypsy moth (Lymantria dispar), the European pinesawfly (Neodiprion sertifer) the diamondback moth (Plutella xylostella),the grapeleaf skeletonizer (Harrisina americana), the potato tuber worm(Phthorimeaea operculella), the black cutworm (Agrotis ipsilon), thecodling moth (Cydia pomonella), the oriental fruit moth (C. molesta),and the navel orangeworm (Amyelois transitella).

Baculovirus production is known to those skilled in the art and occursby fermentation in cultured insect cells (Vlak et. al., 1990, BioreactorDevelopment for Production of Viral Pesticides or Heterologous Proteinsin "Insect Cell Cultures," Ann. N.Y. Acad Sci. 589, 399-418; Taticek etal., 1995, Overview of issues in bioreactor design and scale-up, in:"Baculovirus Expression Systems and Biopesticides," Shuler, Wood,Granados and Hammer, eds. Wiley-Liss, New York), or, in vivo in massreared insect larvae (Ignoffo, 1967, Possibilities of Mass-ProducingInsect Pathogens, Internal. Colloq. Insect Pathol. Netherlands, pp.99-117; Shapiro 1986. In Vivo Production of Baculoviruses in: TheBiology of Baculoviruses, Vol. II, CRC Press; Shieh 1989, Industrialproduction of viral pesticides, Adv. Virus Res. 36, 315-343).

Durable TiO₂ means TiO₂ (either anatase or rutile) that has been madedurable by coating the TiO₂ particles with an oxide coating consistingof alumina, silica, or a combination of the two. An example of such amaterial is "TI-PURE®" Titanium Dioxide Pigment R-706 which ismanufactured by the DuPont Company, Wilmington, Del. It is a TiO₂ onwhose surface is deposited successive layers of silica and alumina whichgreatly reduces the photochemical reactivity of TiO₂. Any combination ofsilica or alumina or both may be contemplated however the preferred isone which has alumina as the outside layer. Preparation of thesematerials is described in Inorganic Pigments, Manufacturing Processes,Chemical Technology Review No. 166, M. H. Gutcho, ed. Noyes Data Corp.Park Ridge, N.J. 1980 and references cited therein. The importantfeature is that the alumina, silica, etc., should form a continuous,nonporous coating on the TiO₂ particles and not be present as a simpleadmixture. Typically these materials contain 1 to 4% silica (as SiO₂)and/or 4 to 9% alumina (as Al₂ O₃), both based on the weight of TiO₂.

The durable TiO₂ can be simply mixed with the virus at a weight ratio of10 to 90% weight/weight (w/w) TiO₂ to virus. Preferably though, thedurable TiO₂ is substantially coated on the surface of the virus.Substantially coated means the TiO₂ is electrostatically fixed to thesurface of the virus particle. Combination of the virus with the durableTiO₂ can occur by any standard means known in the art [J. Econ. Entomol.69, 731(1976)] but is advantageously coated onto the individual virusparticles by the process of the present invention. It is most preferredthat the biological pesticide particles be completely coated asindividual particles or as heteroflocculated particles (i.e., someinterior particles may not be coated or are partially coated, but theoutside of the heteroflocculated particles are completely coated).

According to one preferred embodiment of the present invention, additionof an aqueous slurry of the R-706 durable TiO₂ particles to a stirredaqueous slurry of a baculovirus at neutral pH (typically between about5.5 and 8.0) causes the TiO₂ particles and the baculovirus solids toco-precipitate leaving a somewhat clear layer at the top of the liquidand a dense floc at the bottom. This is not observed with uncoated TiO₂products (anatase or rutile) or TiO₂ products containing anionicsurfaces such as silica-coated or those containing anionicsurface-active agents. By virtue of its alumina coating, the durableTiO₂ particles are positively charged in aqueous suspension at neutralpH whereas the nuclear polyhedrosis virus, and by inference the otherimpurities in the baculovirus, are negative. The existing chargedifference and mutual attraction causes the system to "collapse" andproduces the observed flocculation. This is a very beneficial effect inthat it results in the TiO₂ being electrostatically (as opposed tocovalently) bound to the surface of the virus; thereby providing a veryefficient means of surrounding each virus particle with a reversiblyattachable UV shading material. This material is then spray dried toagglomerate the flocs into particles less than 50 micron mean volumediameter. This step provides resistance to redispersion of the durableTiO₂ away from the virus, which might occur during use in the presenceof aggressive waters or surface active agents commonly employed inagricultural sprays, such as wetters or spreader-stickers whileproviding rapid dispersal under the very alkaline conditions of thetarget insect mid-gut where the pH can be ≧10.5.

Certain particles which have light-filtering attributes but which bear anegative charge may be converted to bear a positive charge in which casethe processes and benefits observed with durable TiO₂ are also observed.For example, a 5% suspension of Raven 430 Carbon (Columbian ChemicalsCo.) containing 0.5% w/w of Ethoquad T/13-27W (Akzo Nobel Chemicals,Inc.) becomes positively charged. This suspension is also capable offlocculation like durable TiO₂ with the virus. Particles which havelight filtering attributes include, but are not limited to TiO₂, carbonand zinc oxide. Any means for converting the particles to bear apositive charge is acceptable, however, coating the particles asdescribed for durable TiO₂ and use of suitable quaternary ammoniumsurfactants are preferred such as Ethoquad.

In commercial operation, the present composition will usually, but notnecessarily, be diluted in an aqueous matrix and sprayed on the locus tobe treated.

The present composition can optionally include inert ingredientstypically found in agricultural formulations such as mineral carriers ordiluents, surfactants to enhance wetting of the spray target, vegetableflours, animal proteins or other gustatory stimulants to encouragefeeding on the formulations, buffering agents to maintain an optimum pHfor the formulation, antifoam agents, and preserving or stabilizingagents to inhibit microbial growth or other degradative processes.

The present composition can also contain up to about 50% w/w certainfluorescent brighteners or stilbenes; or, alternatively, be addedseparately to an aqueous spray solution of the present composition.These materials are well known to provide enhanced activity of a rangeof baculoviruses to their hosts and are active at concentrationstypically ranging from 0.01 to 1% by weight of the spray liquid butuseful at higher concentrations. Suitable stilbene materials aredescribed in U.S. Pat. Nos. 5.124.149 and 5,246,936.

Preferred stilbene compounds are the analogs of4,4'-diamino-2,2'-stilbene disulfonic acid, namely a Blancophor(available from Mobay Chemicals, Pittsburgh, Pa.) such as BlancophorBBH, Blancophor MBBH, Blancophor BHC, etc., a Calcifluor White(available from Sigma Chemical, St. Louis, Mo.) such as CalcifluorWhite. Calcifluor White M2R, Calcifluor White ABT, Calcifluor White LD,Calcifluor White RWP etc., a Leucophor (available from Sandoz ChemicalsCorp., Charlotte, N.C.) such as Lucophor BS, Lucophor BSB, Lucophor EKB,Lucophor PAB, etc.; a Phorwite (available from Mobay Chemicals,Pittsburgh, Pa.) such as Phorwite AR, Phorwite BBU, Phorwite BKL,Phorwite CL, Phorwite RKK, etc., Blancophor BBH, Calcifluor White M2R,and Phorwite AR are the most preferred stilbene compounds.

EXAMPLE 1

This example illustrates the general procedure for producing theflocculated durable TiO₂ /virus mixtures. One hundred grams of durableTiO₂ particles (DuPont TI-PURE® Grade R-706) was bead-milled for 20 minwith 100 g of deionized water. The pH was adjusted during milling to arange of 7.0 to 8.0 with dropwise addition of sodium hydroxide. Theresultant particle size of this positively charged suspension was 0.8microns mvd (mean volume diameter). This slurry was added with highspeed mixing to 500 g of virus [Autographa californica multicapsidnuclear polyhedrosis virus (MNPV)] aqueous slurry (15% w/w suspendedsolids) obtained by infection of Heliothis virescens larvae. The slurryhad been de-oiled by centrifugation. The virus solids typically contain1×10¹⁰ to 1×10¹¹ virus occlusion bodies OBs per gram, the virus used inthis example contained 2.4×10¹⁰ (OBs) per gram dry weight. The pH of theresulting slurry was lowered to 6.5 by dropwise addition of hydrochloricacid and stirred briefly to permit completion of the process. Theresultant mixture was spray dried in a pilot-scale spray dryer at aninlet temperature of 185° C. and an outlet temperature of 60 to 65° C. Atotal of 100 g of durable TiO₂ coated virus formulation was recoverednominally containing 42% (1×10¹⁰ virus OBs per gram). The virusconcentration cannot be directly measured in these systems becauserelease from the matrix is only achieved at pH levels high enough tobegin dissolving the virus particles. The particle size of thespray-dried material was 20.2 microns mvd and an example of a singlecoated virus is shown in FIG. 1.

EXAMPLE 2

This example illustrates the general procedure for preparing a mixtureas in Example 1 with a different virus and the addition of a fluorescentbrightener. Fifty grams of durable TiO₂ particles (DuPont TI-PURE® GradeR-706) and 25.2 g of a stilbene brightener (Blankophor BBH) werebead-milled for 20 min with 75 g of deionized water. The pH was adjustedduring milling to a range of 7.0 to 8.0 with dropwise addition of sodiumhydroxide. The resultant particle size was 1.1 microns mvd. This slurrywas added with high speed mixing to 547 g of virus (Helicoverpa zeaSNPV) aqueous slurry (20% w/w suspended solids). The virus solidstypically contain 1×10¹⁰ to 1×10¹¹ virus OBs per gram, the virus used inthis example contained 5.9×10¹⁰ OBs per gram dry weight. The pH of theresultant slurry was lowered to 6.5 by dropwise addition of hydrochloricacid. The resultant mixture was dried in the same spray dryer as inExample 1 at an inlet temperature of 185° C. and an outlet temperatureof 60 to 65° C. The durable TiO₂ coated virus material was calculated tocontain 5.3×10⁹ OBs per gram. The particle size of the spray-driedmaterial was 18.9 microns mvd.

EXAMPLE 3

This example illustrates the general procedure for preparing a mixtureas in Example 1 with a different virus and the addition of a feedingstimulant. Thirty-five grams of durable TiO₂ particles (DuPont TI-PURE®Grade R-706) and 65 g of corn flour were bead-milled for 20 min with 516g of deionized water. The pH was adjusted during milling to a range of7.0 to 8.0 with dropwise addition of sodium hydroxide. The resultantparticle size was 2.2 microns mvd. This slurry was added with high speedmixing to an aqueous slurry of 500 g of the virus of Example 1(Autographa californica MNPV) slurry (25% w/w suspended solids). Thevirus solids typically contain 1×10¹⁰ to 1×10¹¹ OBs per gram, the virusused in this example contained 2.4×10¹⁰ OBs per gram dry weight. The pHof the resultant slurry was lowered to 6.5 by dropwise addition ofhydrochloric acid. The resultant mixture was dried in a spray dryer asin Example 1 . The durable TiO₂ coated material was calculated tocontain 1.3×10¹⁰ virus OBs per gram. The particle size of thespray-dried material was 26.8 microns mvd.

EXAMPLE 4

This example illustrates the general procedure for preparing a mixtureas in Example 1 with a different virus and the addition of a binder.Twenty-five grams of durable TiO₂ particles (DuPont TI-PURE® GradeR-706) and 10 g of corn starch were bead-milled for 20 min with 75 g ofdeionized water. The pH was adjusted during milling to a range of 7.0 to8.0 with dropwise addition of sodium hydroxide. This slurry was addedwith high speed mixing to 200 g of an aqueous slurry of virus(Autographa californica MNPV) slurry (20% w/w suspended solids). Thevirus solids typically contain 1×10¹⁰ to 1×10¹¹ OBs per gram, the virusused in this example contained 2.4×10¹⁰ OBs per gram dry weight. The pHof the resultant slurry was lowered to 6.5 by dropwise addition ofhydrochloric acid. The resultant mixture was spray dried in a spraydryer as in Example 1. The durable TiO₂ coated material was calculatedto contain 1.3×10¹⁰ virus OBs per gram.

EXAMPLE 5

This example illustrates the field stability achieved using thematerials and process indicated in Example 1. Plots consisted of two 100ft rows divided into four 25 ft replicates. Treatments were applied at20 gallons-per-acre (0.18 m³ per hectare) with a 0.125% w/w aqueoussuspension of unformulated virus (Autographa californica MNPV) using anapplication rate of 2.5×10¹¹ OBs per acre (1×10¹⁰ OBs per gram ofvirus). Another row of cotton was treated in the same manner andocclusion body rate using a 0.66% w/w aqueous dispersion of the powderdescribed in. Example 1. At various time intervals whole cotton leafsamples were taken from each treated row along with an uncontaminatedcheck row. Following application, plants were allowed to dry then 16leaves per replicate was excised for laboratory bioassay. A single leafwas placed into each cell of a 16 celled jelly tray. A single 3-day-oldH. virescens larva was also added to each cell. Test units were held at28° C. in 14 h of light for a total of 5 days. Mortality was assessed onthe fifth day. Repeated leaf collections and bioassays were conducted 1,2, and 4 d following the application. The results shown in Table 1illustrate the stabilization achieved on addition of the durable TiO₂particles.

                  TABLE 1                                                         ______________________________________                                        Comparison of formulated virus and unformulated virus                                   Percent Kill                                                        Sampling Time                                                                             Unformulated                                                                             Formulation in Example 1                               ______________________________________                                        0           58         49                                                     1 day       30         72                                                     2 days      16         56                                                     4 days      6          47                                                     ______________________________________                                    

EXAMPLE 6

This example compares the activity of formulations prepared as inExamples 1 and 3 (with and without spray drying) as well as showing thatthe activity differences are maintained on a different surface (glass)as well as addition of a commonly used agricultural surfactant Tween20®. In this Example, microscope slide coverslips were treated with 10μl of virus preparation containing 1×10⁸ OBs per ml as calculated fromthe amount contained in each preparation and the suspension alsocontained 0.2% w/w Tween-20® surfactant. Half of the treated coverslipswere exposed to natural mid-day sunlight for 4 h and the other half keptin darkness. The virus was stripped from each of the coverslips bysonication (10 s) in 10 ml of distilled water. A molten lepidopterandiet was added to the suspension of each sample and then cast into 2-25well bioassay trays. Each replicate consisted of fifty 3-day-old H.virescens larvae. Mortality assessments were made 144 h post exposure.The percent activity remaining was calculated by comparing UV exposed toUV unexposed virus. This example also illustrates the importance ofspray drying the product prior to use.

                  TABLE 2                                                         ______________________________________                                        Comparison of formulations prepared according to Examples 1 and 3             after 4 hours of sun exposure.                                                Percent Kill                                                                  Example 1  Example 3                                                                              Example 1 w/o Spray Drying                                ______________________________________                                        82         96       17                                                        ______________________________________                                    

What is claimed is:
 1. A pesticidal composition comprising negativelychanged particles of a viral pesticide and positively charged particlesof durable TiO2 in an amount sufficient to substantially coat theparticles of the viral pesticide wherein said durable TiO₂ particleshave a continuous, nonporous coating comprising alumina forming acationic surface on said durable TiO₂ particles, and wherein saiddurable TiO₂ particles are electrostatically fixed to the surface of theparticles of said viral pesticide.
 2. The pesticidal composition ofclaim 1 wherein the viral pesticide is a baculovirus.
 3. The pesticidalcomposition of claim 2 wherein the baculovirus contains genes encodingan insecticidal protein.
 4. The pesticide composition of claim 2 whereinthe particles of baculovirus substantially coated with durable TiO₂ havea mean particle size distribution of less than about 50 μm.
 5. Thepesticidal composition of claim 1 further comprising at least one of asurfactant, a solid diluent or a liquid diluent.
 6. The pesticidalcomposition of claim 5 further comprising a fluorescent brightener.
 7. Aprocess for preparing the composition of claim 1 comprising mixingpositively charged durable TiO₂ particles having a continuous, nonporouscoating comprising alumina forming a cationic surface on said durableTiO₂ particles with an aqueous slurry comprising negatively chargedviral pesticide particles wherein the pH of the aqueous slurry rangesfrom 5.5 to 8, and wherein said durable TiO₂ particles areelectrostatically affixed to the surface on the particles of said viralpesticide as a result of mixing said durable TiO₂ particles with saidaqueous slurry of viral pesticide particles at said pH range, and spraydrying the resulting mixture.
 8. A method of controlling agriculturalpests comprising: applying to the pests or their environment apesticidally effective amount of the pesticidal composition of claim 1.